It is the major aim of this preclinical project, to promote resilience against stress-related somatic and affective pathologies in male and female mice by repeated subcutaneous (s.c.), intranasal (i.n.) or intragastric (i.g.) administrations of heat-inactivated preparations of immunoregulatory "old friends from mud and soil", including Mycobacterium vaccae NCTC 11659, M. vaccae ATCC 15483typestrain and M. aurum DSM 33539, and to understand the underlying mechanisms.

Background: Epidemiological data provide strong evidence for a steady rise in the incidence of many stress-associated psychosomatic disorders in developed countries since the 1950s.(1-10) Although the underlying mechanism is not clear, decreased immunoregulation, resulting from decreased numbers of regulatory T cells (Tregs),(11, 12) is likely to play a role. Furthermore, as less people in rural compared with urban areas suffer from stress-associated somatic and mental disorders,(13-19) it is likely that a reduced exposure, especially during early life,(20) to microorganisms with which mammals co-evolved (=Old Friends), at least in part accounts for immunoregulatory deficits, increased stress-induced inflammation and disease prevalence in modern industrialized urban areas.

Main findings: Consistent with what is proposed by the “Old Friends” hypothesis, we and others have shown that repeated s.c. administrations with a heat-killed preparation of M. vaccae NCTC 11659, an abundant saprophytic “Old Friend” from mud with immunoregulatory properties, is effective in: i) stabilizing the gut microbiome,(21, 22) ii) increasing the percentage of Tregs in mesenteric lymph node cells,(21) iii) preventing stress-induced colitis and proinflammatory cytokine secretion from freshly isolated mesenteric lymph node cells stimulated with anti-CD3 antibody ex vivo,(21) iv) preventing stress-induced aggravation of dextran sulfate sodium (DSS)-induced colitis,(21) v) preventing stress-induced exaggeration of anxiety,(21) vi) preventing stress-induced microglial priming and neuroinflammation,(23-26) vii) ameliorating features of age‑associated microglia activation in the amygdala and hippocampus,(27) viii) preventing negative outcomes of sleep deprivation,(28) and ix) enhancing fear extinction.(29). In extension of these findings and in support of using “Old Friends” not only to prevent but also to treat stress-associated disorders, we recently showed that M. vaccae NCTC 11659 also ameliorates stress-induced anxiety when administered repeatedly via the s.c. route during chronic psychosocial stressor exposure, i.e., after the first psychosocial traumatization has occurred.(30) Own studies further confirm the stress protective effects of M. vaccae NCTC 11659 even when administered via the non-invasive i.n.(30) or i.g.(31) route, respectively. In detail, male mice administered i.g. with M. vaccae NCTC 11659 are protected against: 1) the stress-induced increase in splenic TLR2+ and TLR4+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and TLR4+ monocytes/mononuclear (MO)-MDSCs; 2) the increase in functional splenic in vitro glucocorticoid (GC) resistance typically seen following psychosocial stress in combination with significant wounding (=physical trauma); as well as 3) the stress-induced increase in basal and LPS-induced splenic in vitro cell viability.(31)

In a recent study we further showed that the negative behavioral, immunological and physiological consequences of early life stress (ELA) induced by the maternal separation (MS) paradigm in both sexes, although relatively mild, are to a great extent prevented by subsequent s.c. M. vaccae NCTC 11659 administrations.(32) In extension, own data further show that the catabolic bone turnover and osteoporotic bone phenotype induced by ELA in female sex only was prevented by repeated s.c. administrations of M. vaccae NCTC 11659 (Schimmele et al., under review).

Importantly, the stress-protective effects of M. vaccae NCTC 11659 are not specific for this strain but transferable also to M. vaccae ATCC 15483T.(33) In support for the latter, also repeated i.g. administrations of female mice with M. vaccae ATCC 15483T prior to mating protected the male offspring of these females (i.e. intergenerational effects) from developing enhanced LPS-induced ex vivo cell viability of isolated splenocytes, increased delta cell viability (LPS-stimulated minus basal conditions) of isolated and ex vivo stimulated splenocytes in the absence of CORT as well as functional splenic ex vivo GC resistance as a consequence of CSC exposure during adulthood (Schiele et al., in preparation). As also repeated i.g. administrations of a heat-inactivated preparation of M. aurum DSM 33539 have profound stabilizing effects on the composition of the intestinal microbiome and were protective against the aggravating effects of stress on subsequent DSS colitis (Langgartner et al., under review), the stress protective effects reported for M. vaccae NCTC 11659 and M. vaccae ATCC 15483T seem to generalizable also to other nontuberculous mycobacteria (NTM) species.

Main collaborators: Prof. Dr. Christopher Lowry (University of Colorado, Boulder, USA), Prof. Dr. David Slattery (Goethe University Frankfurt), Prof. Dr. Steffen Stenger (Ulm University Medical Center, Ulm, Germany).

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